Stepone plus real time rcr system

RNA from frozen hippocampus was extracted using QIAGEN RNA kit (QIAGEN, Hilden, Germany), and cDNA was synthesized from 1 μg total RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster city, CA, USA). The amplification reactions were performed with 12.5 μL 2 × SYBR Green PCR master mix (Applied Biosystems), 1 μg cDNA, and forward and reverse primers (100 μM each) using StepOne Plus real-time RCR system (Applied Biosystems). The cycling parameters were as follows: 50 °C for 2 min and 95 °C for 10 min; 1 cycle of 95 °C for 15 s and 40 cycles of 60 °C for 2 min, after one initial step at 95 °C for 30 s and 55 °C for 30 s finally. The primers used were as follows: mouse Npas4, forward 5′-AGCATTCCAGGCTCATCTGAA-3′ and reverse 5′-GGCGAAGTAAGTCTTGGTAGGATT-3′; mouse GAPDH, forward 5′-TGTCAAGCTCATTTCCTGGTATGA-3′ and reverse 5′-TCTTACTCCTTGGAGGCCATGTAG-3′.